Abstract
BackgroundCD33 is genetically linked to Alzheimer’s disease (AD) susceptibility through differential expression of isoforms in microglia. The role of the human CD33 short isoform (hCD33m), preferentially encoded by an AD-protective CD33 allele (rs12459419T), is unknown. Here, we test whether hCD33m represents a loss-of-function or gain-of-function variant.MethodsWe have developed two models to test the role of hCD33m. The first is a new strain of transgenic mice expressing hCD33m in the microglial cell lineage. The second is U937 cells where the CD33 gene was disrupted by CRISPR/Cas9 and complemented with different variants of hCD33. Primary microglia and U937 cells were tested in phagocytosis assays and single cell RNA sequencing (scRNAseq) was carried out on the primary microglia. Furthermore, a new monoclonal antibody was developed to detect hCD33m more efficiently.ResultsIn both primary microglia and U937 cells, we find that hCD33m enhances phagocytosis. This contrasts with the human CD33 long isoform (hCD33M) that represses phagocytosis, as previously demonstrated. As revealed by scRNAseq, hCD33m+ microglia are enriched in a cluster of cells defined by an upregulated expression and gene regulatory network of immediate early genes, which was further validated within microglia in situ. Using a new hCD33m-specific antibody enabled hCD33m expression to be examined, demonstrating a preference for an intracellular location. Moreover, this newly discovered gain-of-function role for hCD33m is dependent on its cytoplasmic signaling motifs, dominant over hCD33M, and not due to loss of glycan ligand binding.ConclusionsThese results provide strong support that hCD33m represents a gain-of-function isoform and offers insight into what it may take to therapeutically capture the AD-protective CD33 allele.
Highlights
CD33 is genetically linked to Alzheimer’s disease (AD) susceptibility through differential expression of isoforms in microglia
These results demonstrate that human CD33M (hCD33M) and human CD33 short isoform (hCD33m) have opposing effects on phagocytosis, providing the first experimental evidence that hCD33m has a gain-offunction role in microglia
U937 cells Previously, we demonstrated that CRISPR/Cas9-mediated deletion of CD33 in human U937 cells leads to increased phagocytosis, which can be complemented with lentivirus-mediated transduction of hCD33M [17]
Summary
CD33 is genetically linked to Alzheimer’s disease (AD) susceptibility through differential expression of isoforms in microglia. The role of the human CD33 short isoform (hCD33m), preferentially encoded by an ADprotective CD33 allele (rs12459419T), is unknown. We test whether hCD33m represents a loss-of-function or gain-of-function variant. The rarer rs12459419T allele correlates with decreased AD susceptibility and enhances exon 2 skipping [6, 7], leading to increased production of a short isoform known as human CD33m (hCD33m; m = minor) [8]. The common rs12459419C allele correlates with increased AD susceptibility and favors production of a long isoform of CD33, known as human CD33M (hCD33M; M = major). One unresolved question is whether hCD33m represents a lossof-function variant stemming from decreased expression of hCD33M or whether increased expression of hCD33m has a gain-of-function
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